The ability of HIV to enter a latent state creates a fundamental obstacle to the eradication of HIV-1 infection by anti-retroviral therapy and has prompted continued interest in developing inhibitors that block the re-emergence of latent proviruses. The goal of this research project is to continue the development of new antiviral leads that target the HIV TAR RNA, and inhibit at the same time reverse transcription and transcriptional elongation from the HIV-1 promoter. Such an inhibitor would be able to block viral replication in both acutely and chronically infected cells and target the reservoir of slowly replicating viruses that persists in the presence of Highly Active Anti Retroviral Therapy (HAART). In the previous funding period, we have identified a new cyclic peptide lead that binds to the HIV TAR RNA with unprecedented activity by expanding peptide chemistry to include un-natural peptidic side chains. The lead peptidomimetic compounds penetrate cellular membranes and inhibit viral replication in primary lymphocytes with activity comparable to nevirapine. We have demonstrated that the antiviral activity coincides with inhibition of both reverse transcription and transcriptional elongation. We now propose to: 1. Optimize the antiviral activity of the cyclic peptide mimetics of the Tat protein to inhibit the function of TARby exploiting additional un-natural peptide side chains and modifications of the peptide backbone 2. Establish the molecular mechanism of action of the peptide leads 3. Generate resistance mutants to fully validate the molecular targets of these anti-viral leads